The Construction Of The Recombinant Plasmid Co-expressing Aβ3-10 Gene Of Alzheimer

ObjectiveAlzheimer disease is one of the most common dementia of the old,which is character with the decline of congnitive function and memory impairment in clinical. AD has affect the patients,live apparentaly.Given the rapid growth of the eldly population of our country,the prevalence of AD is expected to increase.It is investigated that there has been up to 120 million old people of six-plus in our country and 0.5 to 5.6 percent of them suffered with dementia,in which AD accounting for fifty percent.None of the available treatments can prevent the progression of AD. Thus,it is important to research AD.The cause of AD has not been clear.The principal theory of the pathogenesis is the amyloid hypothesis.According to the amyloid hypothesis the increased production of Aβis the initiation of the pathologic process.And other pathologic process behind this.Once Aβespecially Aβ42 over deposit in cerebral would be toxic to neuro and terminate in neurodegeneration.A number of approaches to immune therapy for Aβhave been studied in animal models and found worthy of clinical study Active and passive immunization in AD theoretically increase amyloid clearance via phagocytosis and increased effiux of Aβfrom the brain.Early studies in APP transgenic mice with Aβ42 showed reduction in amyloid pathology,delayed cognitive deficits and improve memory tasks.Clinical studies were conducted on the basis of these encouraging animal model findings and the results of a phase 1 study in 20 patients.Unfortunately 18 of 300 phase 2 study patients(six percent)developed autoimmune meningoencephalitis.Recent research has shown the meningoencephalitis was related to T cell response.Thus,how to decrease the T cell reaction with subunit of Aβis a effective measure.Researchers immuned with N-terminal of Aβcould decrease Aβeffectively and avoid side effects. Although immune therapy of AD suffered difficulties,the developed research take a new hope to the patients.Immunization has not lead to clinical inflammatory and has not obviously impaired the function of CNS.It is showed that the non-methylized CpG motif which is ubiquitous have a strong immunostimulating effect.The artifial oligodeoxyribonucleotide which contain CpG can mimic the function of the CpG motif in the bacterial DNA.It can effectively induce the immunoresponsibility of the body if combined with antigen of the protein-class.The CpG OND has the effect of immunology adjuvant.Moreover researches indicate that prolong the tide by multiple mini-tide can make the antigen ability stronger.So in order to strong the antigen ability in our we adopt the multiple-tide and CpG motif in our experiment.In order to search a more safe and efficient treating project,we constructed plasmids which express Aβ3-10 found in AD and CpG gene and investigate the possibility to make specific vaccine for AD with it.The results will provide foundation for further exploring the mechanism of the elimination of Aβ.MethodsAβ3-10 cDNA was found in GenBank.Aβ3-10 and Aβ3-10-CpG gene segments was added safe nucleotide cozak sequence and enzyme side(upstream-EcoRV enzyme side;downstream-XhoI enzyme side)and NotI enzyme side before CPG sequence. EcoRV enzyme digest plasmid POC57 then combined them with Taq enzyme to construct POC57-Aβ3-10-CpG.eukaryotic expression vector,and then competent bactera-DH5αwas converted.Positive clone was picked and Plasmid was extracted by plasmid extracted box.Enzyme digested and sequenced.Then we picked the right vector,enzyme digested POC57-Aβ3-10-CpG and pcDNA3.1+,puried and combined Aβ3-10-CpG and pcDNA3.1+ to construct pcDNA3.1(+)-Aβ_3-10-CpG and then competent bactera-DH5αwas converted.Positive clone was picked and Plasmid was extracted by plasmid extracted box.Enzyme digested and sequenced.ResultEukaryotic expression vector of pcDNA3.1(+)-Aβ_3-10-CpG was constructed and verified by sequencing.ConclusionThe success of construction of the Recombinant Plasmid Co-expressing Aβ3-10 Gene of Alzheimer and CpG Gene can basically meet the requirement of a nice vaccine, which can provide experimental date for the immunological effect of this candidate vaccine.The result provide foundation for further research of the mechanism of the decrease of senile plaque and developing a new gene vaccine.